This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Hydroxylamine (NH2OH) is an intermediate during the oxidation of ammonia by bacteria such as autotrophic ammonia oxidizing bacteria (AAOB) and methane oxidizing bacteria (MOB). Hydroxylamine is a potent mutagen, and is rapidly converted to nitrite (HNO2) by various enzyme systems, including two unrelated enzymes, hydroxylamine oxidoreductase (HAO) and cytochrome P460, each with a unique a heme at their active sites. Both enzymes are present in the model AAOB, Nitrosomonas europaea, although HAO is much more abundant than cytochrome P460, and supports hydroxylamine oxidation at a much higher rate. Although these enzymes are little direct medical importance (although one of them is present in some opportunistic pathogens), their unique active site structure makes them of considerable interest to all biochemists, and the techniques used to study these enzymes can be applied to enzymes of medical interest. Secondly, HAO is responsible for much of ammonia oxidation to nitrite, a major component of the global nitrogen cycle. While the role of HAO in oxidizing hydroxylamine in vivo appears to be well established, the role of cytochrome P460 is less certain, and may be involved in sequestration or removal of nitric oxide (NO) rather than hydroxylamine oxidation. Despite extensive work on the biochemistry of the HAO of N. europaea, the pathways for HAO export and processing and the mechanism of active site heme cross-linking remain unknown, in large part because the enzyme is essential for the survival of N. europaea and also because the gene for HAO of N. europaea has not been expressed in any heterologous host bacterium. In unpublished experiments, I have placed the HAO gene of N. europaea behind the inducible lac promoter on a plasmid, and introduced the plasmid into two bacterial species. Both Pseudomonas aeruginosa and Escherichia coli could produce the HAO polypeptide as an inclusion body, but the export of the polypeptide into the periplasm and insertion of hemes, including heme P460, did not occur. This suggests that a specific enzyme or transporter, other than that generally used for c-cytochromes, may be involved in HAO maturation. It is interesting to note that, in all bacteria with an HAO gene, including N. europaea, the gene is always followed by a gene for a putative membrane protein of unknown function, which might well be an HAO transporter or maturation enzyme (Bergmann et al 2006). We are currently investigating this possibility by placing the gene into a low copy-number expression plasmid (Novagen's pACYCDuet) together with the gene for HAO, and attempting expression in P. aeruginosa, and E. coli. This will determine if this unknown membrane protein is required and sufficient, along with the general secretory pathway and c-heme processing genes (such as the ccm gene cluster of E. coli) found in most bacteria, for HAO expression. If HAO could be expressed in a heterologous host bacterium, the HAO gene could then be modified by site-directed mutagenesis. In particular, mutant HAO enzymes without the active-site tyrosine could be constructed and characterized biochemically (UV-visible spectroscopy, EPR spectroscopy, hydroxylamine oxidation, NO binding or reduction and redox titrations of hemes).